Identifying Novel Causes of X-Linked Heterotaxy

Abstract

Heterotaxy is a congenital disorder characterized by abnormal arrangement of formation of thoracic and abdominal organs due to errors in embryonic left-right patterning, affecting ~1 in 10,000 live births. These patients exhibit considerable phenotypic heterogeneity, with structural heart defects significantly contributing to poor outcomes. Variants in several genes can disrupt laterality, but ZIC3 variants, primarily identified through targeted sequencing of its coding region, are the only recognized cause of X-liked heterotaxy. This dissertation focuses on a heterotaxy pedigree with four affected males, demonstrating an X-linked inheritance. No coding variant in ZIC3 was identified, leaving the pedigree unsolved for over two decades. Initially, the family’s heterotaxy was hypothesized to be caused by a coding variant in a novel heterotaxy locus on the X chromosome. X-exome sequencing identified a missense variant in GPR101, a gene whose closest phylogenetic relative has been implicated in left-right asymmetry in zebrafish. However, subsequent findings from this study and other research groups suggests GPR101 does not regulate left-right patterning, making the hypothesized GPR101 variant unlikely to be disease-causative. The next hypothesis explored was a non-coding variant in ZIC3, undetected by X-exome sequencing. Whole genome sequencing identified a novel, deep intronic variant in ZIC3, initially hypothesized to trigger the inclusion of an intronic sequence as a pseudoexon during RNA splicing. Further analysis revealed the variant profoundly altered RNA splicing, resulting in the production of several novel ZIC3 isoforms and reduced expression of normal ZIC3 protein. These novel isoforms displayed abnormal function in a variety of in vitro and in vivo assays. This marks the first reported instance of pseudoexon inclusion associated with heterotaxy for any gene and underscores the critical need to expand the scope of variant evaluation beyond mere missense and nonsense variants. The clinical and research field must adapt to assess non-coding variants and to consider alternative disease mechanisms, such as abnormal splicing or dysregulated expression of key left-right patterning genes, in unresolved heterotaxy cases.